Immunotherapies (IT) alone or in combination with standard-of-care (SOC) chemotherapy (CT) have been recently hailed as a potentially effective therapeutic strategy for human hematological and solid malignancies. However, cellular and molecular mechanisms involved in combination therapies are not understood, and a concern exists that SOC-CT may antagonize beneficial effects of IT, which is given in order to up-regulate anti-tumor activity of the patient's immune system. SOC-CT has remained a primary treatment for many cancers, including, e.g., acute myeloid leukemia (AML), and its immunoinhibitory activities are well known. Superimposed on pre-existing cancer-induced immune defects, SOC-CT might interfere with potential benefits of IT. In response to PQ11, we propose to investigate molecular and cellular mechanisms mediated in AML by plasma-derived exosomes, virus-size (30-150nm) vesicles, which carry immunosuppressive signals and inhibit natural killer (NK)-cell and T-cell functions and promote expansion of regulatory T cells (Treg). Our data indicate that SOC-CT is associated with a significant increase of immunosuppressive exosomes in plasma of AML patients. Contrary to expectations, delivery of adoptive IT with NK-92 cells to relapsed AML patients does not lead to improved anti-leukemia NK-cell activity. We hypothesize that strongly immunosuppressive exosomes enriched after SOC-CT antagonize not only endogenous anti-tumor immunity in AML but also adoptively transferred NK-92 cells. In Specific Aim 1 of this proposal, we will test this hypothesis using exosomes isolated from AML plasma before and after SOC-CT to evaluate the mechanisms responsible for inhibition of immune cell functions in vitro. The presence, molecular profiles and levels of immunosuppression mediated by these exosomes will be monitored and linked to the incidence of leukemic relapse. In vitro blocking of inhibitory pathways or neutralization of inhibitory factos carried by exosomes is expected to relieve immunosuppression and restore anti-leukemia reactivity of immune cells. In Specific Aim 2, we will use AML PDX mouse models to in vivo investigate effects of exosomes isolated from plasma on leukemia progression. These leukemic mice will be treated with adoptive transfers of NK-92 cells +/- inhibitors of immunosuppressive pathways operated by AML exosomes and defined as promising in Aim 1 to determine whether silencing of exosome- mediated suppression improves therapeutic effects of IT with NK-92 cells. Selective elimination or silencing of immuno-suppressive exosomes, without interfering with immunostimulatory exosomes, in AML or other malignancies, might provide an effective approach to overcoming SOC-CT-mediated inhibition of adoptive cell therapies and other ITs. In the future, the addition of novel exosome-depleting or -silencing strategies to currently available IT with, e.g., checkpoint inhibitors or engineered T cells, could greatly increase beneficial effecs of these therapies which are likely to be administered in combination with or following SOC-CT